Estradiol increases phosphorylation of the 90 kDa heat shock protein not associated with estradiol receptor in MCF-7 cells in culture.
MCF-7 cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine [( 3H]TA). The heat shock protein (hsp-90) bound to receptor was precipitated with monoclonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Hsp-90 not associated with receptor was similarly purified after isolation with the monoclonal antibody AC88. It was found that estradiol treatment of the cells markedly increased phosphate incorporation in the free hsp-90, without affecting heat shock protein bound to receptor. A 6-fold increase in phosphate content was observed after 10 min incubation of the cells with estradiol. A similar effect was seen after treatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). The calcium ionophore A23187 had no influence on hsp-90 phosphorylation, and treatment of the cells with forskolin to increase the cellular content of cAMP had a reverse effect. A 50% reduction of the phosphate content in the free hsp-90 was observed after 15 min treatment. The observation that estradiol, TPA and forskolin had effect only on hsp-90 not bound to receptor is an indication that the receptor-hsp-90 complex exists in vivo. Time course studies show that the effect of estradiol is non-genomic. Two possible explanations of the results seem to exist. Either estradiol induces an increase in the degree of phosphorylation of hsp-90, or hsp-90 is translocated to the cytosol from a different cellular compartment.
Lahooti
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Department of Biochemical Endocrinology, University of Bergen, Haukeland Hospital, Norway.
Thorsen
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Aakvaag
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D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011960:Receptors, Estrogen / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D006360:Heat-Shock Proteins; D011960:Receptors, Estrogen; D005576:Colforsin; D000001:Calcimycin; D004958:Estradiol; D013755:Tetradecanoylphorbol Acetate